We previously demonstrated that cyclic nucleotide phosphodiesterase can be activated by a thiol protease which is released from a dense lysosomal fraction of rat kidney cortex by extraction with hypotonic media. A second more buoyant population of lysosomes appears to be deficient in protease activity. After purification to apparent homogeniety the protease exhibits a molecular weight of 23,000 and multiple charge isozymes (pI approximately 5.6-6.0). Its substrate specificities and inhibitor sensitivities resemble those of cathepsin L from rat liver. Studies utilizing protease inhibitors provide no evidence that proteases participate in the hormonal regulation of phosphodiesterase in the intact cell.